PROJECT SUMMARY/ABSTRACT The goal of this proposal is to understand how normal developmental programs shape the genetic and epigenetic landscapes of acute myeloid leukemia (AML). AML can occur at any stage of life yet the mutations that cause AML differ between childhood and adulthood, especially when one compares young children to adults. For example, the Flt3 Internal Tandem Duplication (FLT3ITD) mutation is common in adolescent and adult AML, but it is rare in infant and early childhood AML. Likewise, MLL translocations are found in a majority of infant AML, yet they are rare in adult AML. These observations suggest that FLT3ITD may transform adult hematopoietic progenitors more efficiently than fetal progenitors, and MLL translocations may transform fetal progenitors more efficiently than adult progenitors. In preliminary studies, we discovered that Flt3ITD and cooperating Flt3ITD/Runx1 mutations caused hematopoietic stem cell (HSC) depletion and myeloid progenitor expansion in adult, but not fetal, stages of life. FLT3ITD activated STAT5 signal transduction in fetal, neonatal and adult progenitors, yet it did not induce changes in target gene expression until ~2 weeks after birth. The data suggest that fetal and neonatal progenitors are protected from transformation because they are not competent to express FLT3ITD target genes. Either they lack key transcriptional co-activators, or the epigenetic landscape of fetal progenitors suppresses FLT3ITD target gene activation. In parallel studies, we tested whether a tetracycline-inducible MLL-ENL allele transforms fetal progenitors more efficiently than adult progenitors. Fetal MLL-ENL induction caused AML in almost all mice tested. Adult induction did not cause AML in any of the mice tested (now at 6 months follow-up). These results suggest that adult progenitors resist transformation by MLL-ENL much like fetal progenitors resist transformation by FLT3ITD. Further work is needed to understand the cis- and trans-regulatory elements that determine when and how individual mutations are competent to transform. In Aim 1, we propose to precisely characterize the transition from fetal to adult transcriptional programs in developing HSCs and myeloid progenitors using Drop-seq and ATAC-seq technologies. In Aim 2, we propose to test whether Flt3ITD and cooperating Flt3ITD/Runx1 mutations have developmental context- specific effects on gene regulation and leukemogenesis. We will test whether enhancers for Flt3ITD and Flt3ITD;Runx1 target genes have age-specific patterns of accessibility and inaccessibility during development, and whether adult-specific, heterochronic transcription factors are necessary for AML formation. In Aim 3, we will test whether MLL-ENL has developmental context-specific effects on gene regulation and leukemogenesis. If we can understand how normal developmental programs interact with genetic mutations to cause malignancies, it may be possible to target these interactions therapeutically.